Hemoproteins have many important physiological roles, including oxygen transport biosynthesis of neurotransmitters, and metabolism of xenobiotics. Over the last five years we have discovered that the activities of hemoproteins can be altered by a mechanism that involves the cross-linking of the heme prosthetic group to the protein. We have focused our studies on characterizing the structure, mechanism of formation, and biochemical properties of the protein bound adducts formed from free radical mediated alteration of hemoglobin and myoglobin. Of particular interest has been the discovery that the covalent cross- linking of the heme to protein transforms myoglobin to an oxidase. We have recently shown that injection of the transformed myoglobin into cultured fibroblasts leads to cell death, whereas injection of native myoglobin had no effect. It appears that covalent alteration of myoglobin may be a mechanism of eliciting toxicity under conditions where reactive intermediates may be formed, for example in ischemia and reperfusion of tissues. Also in this year we have further extended our studies to include other biologically important hemoproteins. It was discovered that oxidative inactivation of prostaglandin H synthase, a key hemoprotein in the synthesis of prostaglandins, prostacyclins and thromboxanes, is due in part to the covalent alteration of the heme prosthetic group to protein bound products. The protein bound product likely arises from the cross-linking of the heme to an active site amino acid residue. Such a mechanism could account for the inactivation of the synthase observed during the normal catalytic function of the enzyme. In addition we have also found that the neutrophil NADPH oxidase, a multi-protein complex with a hemoprotein component that is an important line of defense against infection, is inactivated by arachidonic acid concomitant with the alteration of the heme prosthetic group. In the presence of a cytosolic protein (p47) the oxidase is protected from heme alteration and inactivation suggesting a biological role for this cytosolic protein. Thus, it appears that a variety of hemoproteins undergo prosthetic heme alteration and that these processes may have biological effects.